PROJECT SUMMARY/ABSTRACT Diabetic gastroparesis is defined by delayed emptying of gastric contents in the absence of mechanical obstruction. The cellular changes that underlie diabetic gastroparesis were, until recently, poorly understood. As a result of our previous work we know that increased oxidative stress, loss of ICC and loss of Nos1 expression are the most common cellular abnormalities in diabetic gastroparesis. We now focus on determining if there is a common unifying abnormality that leads to the diverse cellular changes seen in diabetic gastroparesis. Our overall hypothesis is that in the genetically predisposed, gastric macrophages are central to the development of the cellular damage that leads to gastroparesis. Activation of alternatively activated macrophages that express heme oxygenase-1 (HO1) protects the stomach while activation of classically activated macrophages results in cell damage. We will test the central hypothesis in 3 specific aims. In SA1 we will determine why only a subset of patients with diabetes gets gastroparesis. In SA2 we will determine the molecular mechanisms involved in the development of diabetic gastroparesis and in SA3 we will determine pathways to reverse diabetic gastroparesis. The SAs are supported by strong preliminary data which show: 1. Repeat sequences in the promoter of the human HO1 gene (HMOX1) are associated with diabetic gastroparesis. 2. Diabetic op/op mice that lack gastric macrophages do not develop delayed gastric emptying. 3. Induction of macrophages in diabetic op/op mice by mCSF-1 results in ICC loss and delayed gastric emptying. 4. Media from cultured M1 macrophages induces loss of ICC while media from M2 macrophage cultures protects against damage from M1 media. 5. Mass spectrometry on media from M1 and M2 macrophage cultures has identified key mediators of the effect of the media. 6. Next generation sequencing of human gastric full thickness biopsies has identified key molecules and pathways associated with diabetic gastroparesis. 7. A comprehensive drug screen has identified FDA approved medications that can be used to induce HO1 and treat gastroparesis. 8. Delivery of the top drug candidates and of HO1 by the adeno-associated virus, AAV9 reduces oxidative stress and reverses delayed gastric emptying. We will use Western blots, RT-PCR, single cell PCR, quantitative PCR, next generation sequencing, mass spectrometry, microelectrode recordings, immunohistochemistry, high throughput drug screening, AAV9 virus delivery, RNA knock down techniques, and organotypic and single cell cultures to investigate the central hypothesis. The significance of this work is that, as a result of the work done in the previous grant cycles and the preliminary data presented in this proposal, we will substantially advance our understanding of the cellular and molecular level events responsible for gastroparesis by providing a unifying hypothesis for how diabetes results in damage to key cell types and importantly how targeting macrophages allows the development of, for the first time, disease modifying agents rather than symptom management.